Overall, in the late arousal samples, tau phospho levels had decreased and showed degrees comparable to those of euthermic, non-hibernating animals with slight variations regarding the analysed brain regions. The highest degree of tau phosphorylation was observed in the neocortex and the midbrain, including the hypothalamus. In both regions most of the analysed phosphosites showed hibernation-state dependent reversible tau phosphorylation, whereas the analysis of hippocampus, brainstem and cerebellum revealed a more complex pattern. The tau phosphosite T231/S235 was the only site showing reversible phosphorylation in all brain regions analysed. Interestingly, for this site we also observed aberrant phosphorylation kinetics. In general, tau phosphorylation did not further increase in late torpor. However, phospho levels at T231/S235 were significantly Gomisin-D elevated in late torpor compared to early torpor. This increase was detected in both arctic ground squirrels and Syrian hamsters indicating a conserved characteristic of this particular site. In black bears sampled during hibernation all seven of the investigated phosphosites showed an increased phosphorylation level compared to summer active animals, both in frontal cortex and hippocampus. We applied the Alz-50 antibody that specifically detects a particular tau conformation, typically for PHF-tau. The analysis of protein extracts from both arctic ground squirrels and Syrian hamsters revealed 
no conformational change. In contrast, in the frontal cortex of hibernating black bears we found a significant increase of immunoreactivity. This finding shows that in black bears an elevated tau phosphorylation is associated with conformational changes, which are typically related to pathological alterations in neurons. There was no overall pattern of altered tau protein expression. Nevertheless, tau protein expression was decreased in the brainstem and midbrain of torpid arctic ground squirrels compared to euthermic animals. It was also reduced in the cerebellum of torpid Syrian hamsters. Tau expression reverted to euthermic levels after arousal except for the midbrain of arctic ground squirrels where expression was still reduced in late arousal. There were no changes of tau protein expression in black bears. Regarding tau mRNA isoform expression we focused on the analysis of a potentially altered splicing of exon 10 that encodes for an additional microtubule binding repeat and thereby alters the binding capacity of tau. Most interestingly, there was no general shift towards increased phosphorylation in torpor and arousal. We rather determined a temperature dependent variation in tau phosphorylation in these hibernation states. The most intense phosphate intake was observed at 30uC in torpid animals resulting in an almost doubled phospho-tau degree. To further analyse this temperature dependency, we measured rate of tau phosphate net turnover at a series of temperatures that simulated the entry into torpor and arousing in vitro. Brain samples were taken from euthermic, torpid and aroused animals and subjected to a decreasing and an increasing temperature gradient. As shown in Figure 9, temperature dependency of phosphate incorporation showed characteristic and significant differences between tissues taken from animals at different Mechlorethamine hydrochloride stages during the hibernation cycle. Tissue taken from torpid animals was most sensitive to the temperature shift, followed by tissue from aroused and euthermic animals. In torpid animals, for example, the phospho-tau level was already higher after an incubation time of 12.5 min compared to that of euthermic animals after the total incubation time of 66 min. In ground squirrels, the microtubule-associated brain protein tau enters a hyperphosphorylated state during hibernation that is fully reversed when animals periodically arouse to normal levels of metabolism and brain temperature. Since these changes occur spontaneously and seemingly without pathology.